Macromolecules 1994, 27, 7601-7605
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Molecular and Crystal Structure of the Anhydrous Form of Chitosan Toshifumi Yui* and Kiyohisa Imada Department of Materials Science, Miyazaki University, Miyazaki, 889-21 Japan
Kenji Okuyama, Yutaka Obata, and Katsumi Suzuki Faculty of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184 Japan
Kozo Ogawa Research Institute for Advanced Science and Technology, University Sakai, Osaka, 593 Japan
of Osaka Prefecture,
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Received June 17, 1994; Revised Manuscript Received August 19, 1994s
ABSTRACT: The molecular and crystal structure of the anhydrous form of chitosan, a (l-*4)-linked 2-amino-2-deoxy-/3-D-glucan, has been determined by combined X-ray diffraction analysis and stereochemical model refinement. Chitosan chains crystallize in an orthorhombic unit cell with dimensions a 0.828 nm, h 0.862 nm, and c (fiber axis) = 1.043 nm. Systematic absences are consistent with the space group P2i2i2i. The unit cell comprises four glucosamine residues, giving a density of 1.52 g/cm3. The X-ray diffraction pattern was recorded on the imaging plate, and an intensity of each reflection spot was estimated by two-dimensional measurement and subsequent background removal. Chain conformation is an extended 2-fold helix stabilized by intramolecular 03’ -05 hydrogen bonds, and an 06 atom is rotated at near gt position. Two chains pass through the unit cell with an antiparallel packing arrangement. Intermolecular N2- -06 hydrogen bonds contribute to the three-dimensional stabilizing of the crystal structure. The reliability of the structure analysis is indicated by the X-ray residual R = 0.175 and R" 0.178 against the observed 38 hkl’s. Some significant similarities are observed between the anhydrous crystal structures of the present chitosan and (1—4)-/3-D-mannan. =
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Introduction
These chitosan polymorphic forms correspond to the hydrated crystals in which the water molecules may or may not be located at the crystallographically defined positions in the unit cell. Upon heating the chitosan samples in water, the crystals were converted into the anhydrous form,10-11 which was considered to be the simplest polymorphic form containing no small molecules, such as water and metal ions. It has been also observed that chitosan samples rich in anhydrous crystals become insoluble in acidic solvents in which those involving the noncrystalline region and hydrated crystals are easily dissolved.11 Such physical properties of chitosan, arising from the crystalline polymorph and the crystallinity, might be correlated to its chemical and biological activities. The present work was undertaken to determine the detailed crystal structure of the anhydrous form of chitosan. Given knowledge of the molecular and packing structure of the chitosan chains in the crystal, the crystal structures of more complicated as well as more interesting polymorphs of chitosan, such as the hydrate,3 metal complexes,2 and salts,4 will be determined in future studies.
Chitosan is a linear polymer of /3-(l-*4)-linked 2-amino2-deoxy-D-glucose residues. It is readily prepared from chitin by chemical N-deacetylation and, as well as chitin, has been considered a promising material for various industrial applications. Having a regular distribution of aliphatic primary amino groups, chitosan exhibits a remarkable ability to form complexes with transition metals and salts with some acids from aqueous solution. We have reported the behavior of chitosan-metal complex formations using X-ray diffraction measurement.1’2 It has been observed that an extended 2-fold helical conformation of the chitosan chain, originally found for tendon chitosan,3 is retained in most of these crystals. Other X-ray diffraction studies on some chitosan salts (HF, HC1, and H2SO4 salts)4 and chitosan gel5 revealed that the chitosan backbone adopted either a left-handed (85) or right-handed (83) helix conformation. This type of helical structure was supported by the high-resolution solid-state 13C NMR spectroscopy measurement of the chitosan salts, which suggested the presence of a 4-fold helical conformation of dimeric units.6 As regards interactions between chitosan chains and metal ions in the complex crystals, two types of coordination mode have been proposed: the “pendant model”1’7 and the “bridge model”.8’9 In the former model, a metal ion is coordinated to a single nitrogen atom like a pendant, whereas a metal ion in the latter involves four nitrogen atoms to form a “bridge” between the amino groups of intra- or interchains. A detailed crystal structure analysis shouild be undertaken by analyzing the X-ray diffraction data in order to resolve such ambiguities. ®
Abstract published in Advance ACS Abstracts, November
Experimental Section Nomenclature. Figure
shows the principal parameters
conformation usually prefers the three staggered positions, each of which is referred to as either gauche—trans (gt), gauche—gauche (gg), or trans—gauche (tg).12 Typical values
1,
1994.
0024-9297/94/2227-7601$04.50/0
1
to describe chitosan chain conformation together with the atom labeling. The rotations of the two glycosidic linkages, 4> and T, are defined by the four-atom sequences 05—C1-01-C4' and Cl—01—C4'—C5', respectively. Coupled with these rotations, the glycosidic bridge angle, r, is allowed to vary in the course of conformational refinement of the chitosan chain. The orientations of the primary hydroxymethyl groups around C5— C6 bonds (p is defined by the 05-C5-C6-06 sequence. The
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1994 American Chemical Society
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Macromolecules, Vol. 27, No. 26, 1994
Atomic labeling scheme and designation of the conformational angles d>, T, and %
Figure
1.
of x for the respective positions are gt = 60°, gg = —60°, and tg = 180°. Materials. A tendon chitosan was prepared from the chitin crab tendon, Chionecetes opilio O. Fabricus, by N-deacetylation with 67% sodium hydroxide solution at 110 °C for 2 h under nitrogen atmosphere, following by treating with 1 M aqueous NaOH and subsequently washing with water.2 This solid-state deacetylation procedure was repeated twice. It was revealed by measurement of a colloidal titration that the degree of N-acetylation of the resultant tendon chitosan was near 0%.n The chitosan sample showed the diffraction pattern typical of the hydrated polymorphic form when X-rayed. Its crystallinity was improved by annealing in water at 240 °C in a sealed bomb, and simultaneously, the crystalline structure was converted into the anhydrous form. X-ray Measurement. A rotating-anode X-ray generator (RU-200, Rigaku) was operated in normal focus mode (focus 0.5 x 10 mm2) to provide a monochromatized X-ray beam (Cu Ka, A = 0.154 18 nm, 50 kV x 140 mA). Diffraction data were recorded on the disk-shaped imaging plate (Fuji), a flexible plastic plate coated with fine photostimulative phosphor crystals, with a sample—plate distance of 75.5 mm. The diffraction pattern was read by measuring the fluorescence intensity stimulated by a focused He-Ne laser beam that scanned spirally the surface of the imaging plate. The representative diffraction pattern recorded on the imaging plate is shown in Figure 2. The measurement of X-ray diffraction data was implemented by the hardware system, DIP100S (MAC Science). The intensity values thus obtained converted into pixel data in a rectangular coordinate system. A whole area of the imaging plate (200 mm diameter) was covered with 1600 x 1600 pixels, each having a size of 125 pm2. Removal of background intensity was carried out separately at each diffraction spot. The background surface was estimated by smoothly connecting the intensity levels at the four comers of a fan-shaped area that contained a single or more peaks. The intensity of each diffraction was then subjected to corrections for the Lorentz and polarization factors.
Structure Analysis and Refinement. The structure determination and refinement were carried out using the linked-atom least-squares program (LALS).13 Models of molecular and packing structures are established by minimizing a quantity, Q, defined by the expression O
=
z
where, in the first term,
-
k2\K.f) + 2fy + Fm,0
and
F„,tC are
X kqGq
(1)
the observed and
2. Fiber X-ray diffraction pattern of the chitosan anhydrous polymorphic form recorded on an imaging plate. The fiber axis is vertical, and the calibration line is the d = 0.2319 nm line of NaF.
Figure
calculated amplitudes, respectively, k is a scaling factor, and m counts all individual reflections. The weight, wm, was equal to 1.0 for all the observed reflections, 0.5 for the unobserved ones for which Fmx > F,„„, and 0, for Fmx < Fm,0. The second term evaluates the nonbonded repulsion arising between the nonbonded atoms i and j. The interaction is applied only when the distance between them falls within the standard value. The third term involves a set of coordinate constraint equations, together with their initially undefined Lagrange multipliers, A,. The constraints usually are used to preserve a helix continuity and a ring closeure of the residue during chain of conformational refinement.
Results In our previous report,10 it was suggested that the unit cell dimension of anhydrous chitosan was orthorhombic with a 0.824 nm, b = 1.648 nm, and c (fiber = nm. The unit cell contained eight glu1.039 axis) cosamine units, corresponding to four chitosan molecular chains of a 2i helix conformation. The calculated crystalline density, pcaicd 1.52 g/cm3, was in reasonable agreement with the observed one, p0bsd = 1.44 g/cm3. Absences of (odd,0,0) and (0,odd,0) reflections indicated the possible presence of a crystallographic 2i axis along both the a and b axes of the unit cell. However, most of the 22 individual reflection spots detected in the diffraction data could actually be indexed with a halfsized, two-chain unit cell about the b axis. Only the third layer’s reflection (0.34 nm) with very weak intensity was unable to be assigned, and this caused doubling of the base plane dimension. The fiber diffraction data obtained in the present study exhibited similar quality in crystallinity but were higher in orientation compared with the previous data. Along with an improvement of the diffraction pattern, it was also observed that the 0.34 nm reflection on the third layer disappeared as the annealing temperature was raised to 240 °C, which as a result allowed us to adopt the two-chain unit cell. In a practical viewpoint, the structure determination can be more conveniently carried out by adopting a small unit cell. Inside the observed diffraction region, the four-chain unit cell yielded 29 unobserved hkl’s out of a total of 68 hkl’s, while halving the base plane size decreased this number to 7 out of a total of 40 hkl’s and consequently reduced ambiguity in the structure deter—
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Structure of Anhydrous Chitosan
Macromolecules, Vol. 27, No. 26, 1994
Table
1.
Comparison of Calculated and Observed d-Spacings
d-spacing (nm)
d-spacing (nm)
hkl
calcd
obsd
hkl
calcd
obsd
110 020 200 120 210 220 130 310
0.5971 0.43101
0.583
002 012 102
0.5215 0.44621
0.520
112 022
0.3928 0.33221
202 122 212 222
0.3242/
011 101 111
0.66441
121 211 221 031 301
0.4140] 0.38231
0.3732] 0.2986 0.27151 0.2629J
0.6485/ 0.5182 0.35901
0.3514/ 0.2870 0.27701
0.2668/
0.417 0.372 0.288
0.264
0.650 0.501
0.348 0.280 0.262
0.4413/
0.30831
0.3035/ 0.2591
013 103 113 023 203 123 213
0.32241
014 104 114
0.24961
0.3206/ 0.3005 0.27061
0.2662/ 0.25721
0.2544/ 0.2487/ 0.2390
0.437
0.386 0.319
7603
calculated for the hk0 data at each position. For the P2i2i2i space group, the curves of the residuals exhibited the four minima at fi —150°, —120°, —60°, and -30°. The behavior of the crystallographic residuals at the former two minima were essentially identical to the latter two since interchanging between the a and b dimensions (an operation equivalent to rotating the helix axis 90°) caused little change in the base plane =
projection under the symmetry. The values of the residuals were as follows: R = 0.08, R" = 0.09 for u -20° or -120°, andf? = 0.10, R" = 0.11 for fi -60° or -150°. The curves calculated for the P2i22i space group consisted of the two distinct minima at /i = -120° [R = 0.29, R" = 0.30) and -150° (R = 0.35, R" = 0.31) along with the two shallow minima with unacceptable values of the residuals (> 0.60). Chain translational positions, w in fractions, along the c dimension were determined in a fashion similar to the previous search of /a; w was stepped from -0.25 to 0.25 in 0.05 increments, and the residuals were calculated for all the hkl data, while the chain rotational position, ju, was fixed at either the -150° or -120° position. It was observed that variations of the residuals were relatively insensitive with respect to the stepping of w compared with the residual about u. The minima were detected at w = curves -0.25, 0.0, and 0.15 in the X-ray residual curves calculated for the P2\2\2i space group and w = -0.10 and 0.15 for the P2\22\ space group. Combination of every minima of u and w, coupled with the three staggered positions for the 06 atom rotation, %, provided the 27 initial packing models with the P2i2i2i space group and the 18 models with the P2i22i space group. All the initial models were subject to the chain-packing refinement against the X-ray data by minimizing the first term of the eq 1. After selecting some acceptable models, they were followed by complete structure refinement which involved the simultaneous adjustment of the chain conformational and packing parameters along with variation of the attenuation factors, A. In this step, the total terms of the eq 1 were optimized. Finally, intermolecular hydrogen bonds were introduced as appropriate oxygen atoms were found to be within 0.18-0.32 nm. Table 2 lists the crystallographic residuals, the contact a that represents the stereochemical energy, the packing parameters, and the hydrogen bonds given in the final structures of the best six models with the P2i2i2i space group and the best one with the P2i22i space group. In comparison with the P2i2i2i models, it is apparent that the P2i22i model is unacceptable in terms of both the X-ray crystallography and the stereochemistry. Among the P2\2i2\ models, those with the 06 gt atoms were favored by the X-ray residuals, while rotating the 06 atoms into either gg or tg position quickly raised the values. The same scheme of an intermolecular hydrogen bond (06* *N2 atoms) was detected in each of the P2\2\2i models except model 6 with the 06 tg atoms. Model 1 exhibited the lowest values of R and R" factors together with the lowest contact o and model 2, the second lowest ones. Differences in the X-ray residuals between the best two models are rather small but not insignificant. It, therefore, is quite reasonable to judge model 1 to be the most probable model for the crystal structure of the anhydrous chitosan. The final values for the conformational parameters of the model are = —98°, W -148°, r = 116.7°, and % = 59°. The atomic coordinates in the crystallographic repeat of this model are given in Table 3, and a comparison of the calculated and observed structure factor amplitudes is given in Table 4, showing generally good agreement. Projections of the —
0.298 0.254 0.317 0.298 0.262 0.251
0.247 0.237
mination. Furthermore, systematic absences of (odd,0,0) and (0,odd,0) reflections were still indicated in the equatorial data indexed with the two-chain unit cell. A further refinement of the unit cell dimensions gave the new set of the parameters a = 0.828 nm, b = 0.862 nm, and c = 1.043 nm. A comparison between the observed and calculated d-spacings is listed in Table 1. The presence of a 2i axis on the base plane indicates an antiparallel packing of molecular chains in the crystals, where all the chains pass through the unit cell in alternative direction. This is consistent with the fact that molecular chains of the tendon chitin has also been found to crystallize in an antiparllel chain packing fashion,14 otherwise one must assume an entire rearrangement of the chain polarities to occur during solidstate deacetylation of the tendon chitin to provide parallel chain packing in the chitosan crystals. Possible space group that is content with antiparallel packing of the 2i helix chains, coupled with involvement four glucosamine residues, is either P2i2i2i or the P2i22i if the orthorhombic system is assumed. In both the cases, the molecular chains are placed on the base plane of the unit cell such that the helix axis coincides with a crystallographic 2\ axis along the c, which are at (a/ 4,0) for the both space groups. Thus, the following crystal structure analysis was carried out by assuming the P2i2i2i symmetry and, for comparison, the P2i22i symmetry. A conformational model of the chitosan chain was determined with the constraints of a 2i symmetry and the fiber repeat distance of 1.043 nm. The parameters changed during the structure refinement were those to describe the glycosidic linkage structure: O, W, and r. The sum of the nonbonded interactions that derived from the last two terms in the eq 1 was minimized. In common with other /3-1—1-4-linked glycans of a 2i helical structure, the resulting conformational model involved an intramolecular hydrogen bond interaction between 03 and 05 atoms across every glycosidic linkages. The first stage of the crystal structure analysis was to search appropriate chain-packing positions in the unit cell against the X-ray data. Chain-packing models were first determined about the chain rotational position, fi in degrees, with respect to a or b of the base plane. The helix axis was rotated from -180° to 0° in 20° increments and the crystallographic residuals R and R" were
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Macromolecules, Vol. 27, No. 26, 1994
Table 2. Comparison of Structural Features of Models Selected for Final Structure Refinement j
i
model
X-ray residuals0
06 position
packing parameters 7a 77 w,b frac
fi,a deg
gt gt gt gt
-0.122
tg
-147 -115 -102 -157 -150 -121
0.226
0.175 0.178 0.198 0.213 0.234 0.228
gt
-136
-0.114
0.335
rve
1
2 3
4 5 6
gg
con ac
77’ R
d R
H-bonds® and
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